In silico and in vitro potentials of crocin and amphotericin B on Leishmania major: Multiple synergistic mechanisms of actions

A significant barrier to optimal antileishmanial treatment is low efficacy and the emergence of drug resistance. Multiple approaches were used to monitor and assess crocin (a central component of saffron) mixed with amphotericin B (AmpB) potential in silico and in vitro consequences. The binding behavior of crocin and iNOS was the purpose of molecular docking. The results showed that crocin coupled with AmpB demonstrated a safe combination, extremely antileishmanial, suppressed Leishmania arginase absorption, and increased parasite death. This natural flower component is a robust antioxidant, significantly promoting the expression of the Th1-connected cytokines (IL12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (Elk-1, c-Fos, and STAT-1). In comparison, the expression of the Th2-associated phenotypes (IL-10, IL-4, and TGF-β) was significantly reduced. The leishmanicidal effect of this combination was also mediated through programmed cell death (PCD), as confirmed by the manifestation of phosphatidylserine and cell cycle detention at the sub-GO/G1 phase. In conclusion, crocin with AmpB synergistically exerted in vitro antileishmanial action, generated nitric oxide and reactive oxygen species, modulated Th1, and Th2 phenotypes and transfer factors, enhanced PCD profile and arrested the cell cycle of Leishmania major promastigotes. The main action of crocin and AmpB involved wide-ranging mechanistic insights for conducting other clinical settings as promising drug candidates for cutaneous leishmaniasis. Therefore, this combination could be esteemed as a basis for a potential bioactive component and a logical source for leishmanicidal drug development against CL in future advanced clinical settings.


Introduction
Leishmaniasis is a neglected and intricate transmittable illness produced by Leishmania species and transmitted by female phlebotomine sandflies.It is a zoonotic disease with significant morbidity and mortality, commonly found in humans, rodents, and canines [1][2][3].This disease has various clinical presentations ranging from benign or mild symptoms to non-healing chronic form and ultimate death if left without treatment.Visceral leishmaniasis (VL) is the most deadly and systemic form [4], while cutaneous leishmaniasis (CL) is the furthermost type found in urban (dry) and rural (wet) natures universally.World Health Organization has considered the disease one of the six major tropical diseases endemic in 101 countries and territories where approximately 80% of the burden exists in the Eastern Mediterranean Region [5,6].
During blood feeding, the sandflies inoculate the infective stage or stationary phase promastigotes into the host's body.Next, macrophages phagocytize metacyclic promastigotes in the skin, transforming them into amastigotes (Leishman bodies).Amastigotes proliferate in phagocytes and affect various tissues, producing different clinical forms of leishmaniasis depending upon tissue specificities.Sandflies become infected when they ingest infected macrophages bearing multiplicated amastigotes.The parasites evolve into promastigotes, proliferate into metacyclic forms, and move to the foregut once they reach the midgut.macrophages are the primary cells where Leishmania reproduces and lives for extended durations [7].
A protective immunological response against Leishmania species is initiated mainly by Th1 cell phenotypes, which are vital for inducing macrophage oxidative stress machinery, and reactive oxygen species (ROS), resulting in parasite death.In contrast, a primary Th2 response is harmful to the patient.In this situation, arginase (ARG) and inducible nitric oxide synthase (iNOS) signify two possible immune response pathways.While the former is critical for its development, the latter control parasite growth [8,9].
Presently, leishmaniasis has no effective vaccines, and the available first and second-line treatment choices alone or dual are inadequate.They are associated with severe adverse effects, high cost, long-duration treatment, recrudescence, and Leishmania resistance [13].Other biological control strategies using chemicals are not eco-friendly and unfeasible.Plant-derived products mixed with already available medications have been demonstrated in recent research to be an innovative and synergistic method of treating leishmaniasis.As numerous plant constituents possess immunomodulatory, antimicrobial, and anti-protozoal effects, within normal limits toxicity, they can be considered a substitute medicine source for treating communicable diseases in endemic countries [14].Certainly, several steps including safety, efficacy, and standardization are essential before being registered and marketed.Furthermore, elucidating the mode of action of natural constituents can significantly advance drug development strategies.
Crocus sativus L. is a herbaceous flowering plant in the Iridaceae family that blooms in the fall, contains the red stigma (or saffron), and is extensively cultured in Iran and to a minor extent in Greece, India, Spain, Morocco, Greece, Italy, France, Azerbaijan, and China, [15][16][17].Over 150 active components from saffron have been isolated and available for commercial and medical applications.Antioxidant and anti-inflammatory properties of saffron have also been studied and confirmed for use as drugs that combat depression, malignancy, and other cardiovascular and neurodegenerative conditions [18,19].In traditional medicine, saffron promotes blood circulation, removes blood stasis, and relieves [20].Polyphenols comprising flavonoids abundant in saffron include hesperidin, quercetin, rutin, luteolin, and bioflavonoids.The three critical components of saffron are crocin, which gives the yellow pigment from the stigmas; picrocrocin, which accounts for the corroded, bittersweet taste; and safranal lends the earthy smell to the spice.Crocin is one of the essential alkaloid ingredients of saffron and has many beneficial effects on health.Anticancer, antioxidant, memory, and learning skills properties and increased blood flow in choroid and retina, antimicrobial, and antidiabetic have been demonstrated in many studies [21][22][23].
AmpB is a polyene macrolide used to treat leishmaniasis among various conventional formulations.The discerning activity of this antibiotic against Leishmania species is due to its superior affinity targeting ergosterols biosynthesis, which is more principal in the plasma membrane of these parasites than the cholesterol existing in the mammalian cell membranes [24].Combining different drugs with various mechanistic actions can provide potential synergism, hence exerting a more significant therapeutic effect in treating disease, and this signifies the most promising approach for developing novel leishmanicidal preparations [25].
The previous investigation revealed that crocin possesses an inhibitory effect on the stages of Leishmania major [26].This study aimed to explore crocin and AmpB on L. major stages as a model drug employing a broad panel of in silico-based and preclinical experimental assays.We precisely targeted multiple in vitro approaches to monitor inclusive mechanisms of actions, including molecular modeling, leishmanicidal impacts, safety index, arginase activity, antioxidative and apoptotic values, gene expression fingerprints, and cell cycle profiling.

Study of physical pockets of iNOS protein area.
Measurement of open superficial extents on 3-dimensional (3-D) constructions is essential in advanced experimentations.Molegro Virtual Docker software (Molegro 2011) detected pockets on surfaces and cavities.
Antioxidant activity assessment.To assess the antioxidant properties of crocin, the drug was combined with butylated hydroxyanisole (BHA) in a microtube and 2.6 mL of α, α-diphenyl-β-picrylhydrazyl (DPPH) was included in the mixture.The absorbance (optical density; OD) was then calculated with a spectrophotometer at 518 nm.To determine the essential scavenging action, the absorbance values of the samples were measured and used to calculate the percentage of inhibition [30].
Parasite and macrophage cultivation.The murine macrophage J774-A1 cell line and the standard L. major strain (MRHO/IR/75/ER) in stationary phase were attained from the Kerman Leishmaniasis Research Center in Iran.The culture medium of parasites was RPM 1640 and the macrophage cells were DMEM.All media were enriched with 10% FBS and 1% penicillin G and streptomycin.The study was approved by the Medical Ethics Committee of Kerman University of Medical Sciences.The Ethic approval Code is IR.KMU.REC.1396.2155.
Anti-promastigote assay.To assess the impact of crocin, AmpB, and their combination on L. major promastigotes, an anti-promastigote assay was performed.10 6 cells of L. major promastigotes per mL were counted and cultured on a 96-well plate and treated with 20 μl of several concentrations (0, 12.5, 25, 50, and 100 μM) of drugs.Each concentration was repeated in triplicate.Plates were kept at 25±1˚C for 72 h, then 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide (MTT) was put in each well and incubation continued for 3 h, centrifuged at 3000 rpm for 8 min, and Dimethyl sulfoxide (DMSO) replaced with content, and the OD was measured at 490 nm using a Multi-Mode ELISA (ELX-800-BioTek).The 50% inhibitory concentration (IC 50 ) rate was determined using the SPSS package.
Anti-amastigote assay.To evaluate the effect of crocin, AmpB, and their combination on L. major amastigotes, 1×10 5 /mL of J774 murine macrophages in cultured in 6-chamber slides (Lab-Tek, Nalge Nunc NY, USA) and kept at 37±1˚C in 5% CO 2 for 12 h, then the macrophages infected with 1×10 6 /mL of the metacyclic form of promastigotes and incubated for the next 24h (macrophages to parasite ratio at 1:10).After incubation, free parasites and old medium were removed and changed with new fresh medium and 50 μL of 12.5, 25, 50,100 μM of crocin, AmpB, and combination were added to macrophages.After 72 h we used Giemsa stain to prepare the slide counting the amastigotes under a light microscope [31].
Cytotoxic effects.To assess the potential cytotoxic effects of crocin, AmpB, or their combination, on the J774 murine macrophages, 5×10 5 cells/mL were counted and grown with varying concentrations (0-100 μM) of drugs in 96-well plates.Macrophages were incubated for 72 h at 37 ±1˚C with 5% CO 2 .The untreated for 72 h to evaluate the cytotoxic activities of crocin, AmpB, or in combination.The well included culture and macrophages without medicines as an untreated control group.After incubation, a similar amount of the MTT solution as the anti-promastigote assay was used and incubated for an additional 3 h.The OD was read similarly by the ELISA at 490 nm following adding DMSO solution.SPSS software and a probit test were used to calculate the cytotoxic activity at 50% (CC 50 value).The safety of the drugs was evaluated using the selectivity index (SI), by the following equation (SI = IC50/CC50 � [32].The combination index (CI) was calculated to evaluate the potential synergy of crocin and AmpB.The formula used for CI calculation was as follows: CI = (D)/(Dx) i +(D)/(Dx) ii , where (Dx) i and (Dx)ii show the concentrations of crocin and AmpB and (D) represents a combination of crocin and AmpB.The CI value was used to quantitatively define the degree of synergism (CI<1), additive outcome (CI = 1), or antagonism (CI>1) among the two medications.The theoretic IC 50 was also determined to assess the synergistic activity of the combination therapy.The theoretic IC 50 was calculated using the following formula: theoretic IC 50 = (IC 50 AmpB/2) + (IC 50 crocin/2).
Assessment of arginase level.The activity of arginase in intra-macrophage L. major amastigotes was measured according to the supplier's protocol (Sigma-Aldrich 1 , USA, cat.No. MAK112).Several concentrations of crocin, AmpB, or a combination of both were used to treat the infected macrophages.For this measurement, 1×10 6 intra-cellular amastigotes were lysed with Tris-HCl (10 mM, pH 7.4) and Triton X-100 (0.4%).After that10 mM MnCl 2 was added to the mixture and maintained at 56˚C for 10 min.After centrifugation, 40 μL of supernatant was mixed with 5X substrate buffer and kept for 3h at 37˚C.Finally, 200 μL of urea was used to stop the enzymatic reaction at 25±1˚C.The arginase activity was computed at 430 nm employing an ELISA reader as follows: Gene expression analysis.The comparative expression levels of genes were determined using quantitative polymerase chain reaction (qPCR).RNA was removed from harvested cells using a Qiagen RNeasy mini kit was used to extract RNA, after determining its concentration using a NanoDrop spectrophotometer, a TaKaRa cDNA kit was used and qPCR was performed using an SYBR Green experiment in the Corbett Rotorgene 3000 cycler system.The primers and control gene sequences are given in Table 1 [33].The expression of target genes was evaluated using the 2 -ΔΔCT method, and the ΔCT was calculated using the following formula: [ΔCT = CT(target)-CT(control)].
Determination of NO generation.We use the Griess reaction assay to determine secreted nitric oxide amounts in intra-macrophages amastigotes treated with several concentrations (0-200 μM) of crocin, AmpB, and co-administration.At first J774 murine macrophages (10 5 cells/mL) were infected by L. major parasite (10 6 cells/mL) (macrophage to parasite ratio at 1:10).Cells were kept overnight at 37 ˚C and 5% CO 2 , after that the culture was refreshed and 10 μL of drug added to each well and incubates for 72 h.After incubation, suspensions in each well were collected and NO release was determined colorimetrically in infected macrophages by the Griess reaction.Briefly, supernatants were composed with LPS, and 100 μL of liquid were incubated with an identical size of Griess reagent (Sigma-Aldrich 1 , USA) (1% sulfanilamide, 0.1% naphthyl ethylenediamine, and 2.5% H3PO4) were added into wells kept for 0 min.An ELISA reader then measured the absorbance at 540 nm (Bio Tek-ELX800) [33].
Assessment of reactive oxygen species (ROS).L. major intra-cellular amastigotes treated with crocin, AmpB, and combination.After 24 h, the cells were washed away with PBS (pH 7.4) and overloaded with 10 μM of a permeate probe diacetate 2 0 .7 0 -dichlorofluorescein (Sigma-Aldrich 1 , USA) was diluted in DMSO and incubated at 37±1˚C in a 5% CO2 for 25 min.ROS was measured with the flow cytometer (BD Biosciences) Cell cycle examination.Promastigotes were treated with serial concentrations of crocin, AmpB, or co-administration and incubated at 25±1˚C for 72 h.Then, samples were collected in 1 mL of PBS, fixed with absolute methanol, centrifuged, resuspended at 50 μl of RNase (1 mg/mL), and kept in RT for 30 min after that 1 mL of 0.1 mg/mL of propidium iodide.The DNA content was examined by flow cytometry (Becton Dickinson), and the Cell Quest software assessed the proportion of organisms in different cell phases.
Annexin V/PI and flow cytometry.The assay was undertaken to detect apoptosis of L. major promastigotes treated with different concentrations of crocin, AmpB, or combined using PE Annexin V Apoptosis Detection Kit I (BD Pharnigen ™).Therefore, 1×10 6 promastigotes were cultured in a 1.5 mL microtube.Then, 100 μl of various drug concentrations were incubated at 25±1˚C for 72 h.After that, the organisms were washed with PBS and maintained in a 1mL binding buffer.Then 100 μl solutions were transferred to a 5-mL tube and 5 μl 7-AAD stains plus 5 μl Annexin V was added to each tube and allowed in the dark at 25±1˚C for 15 min.Subsequently, samples were evaluated in a flow cytometer.

Statistical analysis
Analyses were performed by SPSS v. 22.00 (Chicago, IL, USA) and GraphPad Prism v. 8.0 (CA, USA).One-way ANOVA was used to compare the statistical difference among concentrations of drug and paired t-test was completed to find any statistical difference groups.P < 0.05 was set as a significant level.The data of one-way ANOVA test shows in S1 Dataset.

Evaluation of the competence of crocin for binding to iNOS
Lys14, Met135, and Leu136 were predicted as suitable amino acids in mutable residues in catalytic pockets and access tunnels.

Predicting structural protein area
The useful activity of the iNOS protein surface is presented in Fig 1A .We evaluated the 2-D interaction diagrams showing hydrophobic interaction and hydrogen bond necessary energy of the ligand and target protein.We use Ligand Interaction Profiler (PLIP) online server to evaluate the interaction between iNOS and crocin metabolites.Regarding steric relations, crocin interacts with Ser133, Gln134, and Lys13 amino acids of iNOS, respectively (Fig 1B).
Our molecular docking outcomes show crocin binds to iNOS (Fig 1C ), and amino acids active site THR, SER, GLN, GLN, ARG, TRP, TYR, and ASN are active site residues (Table 2 and Fig 1D).The results show that the MolDock score was -241.053kcal/mol.

Effect of crocin on antioxidant action
The compounds ' hydrogen donations evaluated the fundamental scavenging activity of crocin and BHA on DPPH (Fig 2).The effect was a concentration-effect outcome.There was no significant difference between the crocin antioxidant action and BHA's.It shows that crocin has potent antioxidant activity.

Effect of crocin, AmpB, or combination on promastigotes mortality
The mortality profile of treated L. major promastigotes is accessible in Fig 3 .The CC 50 results showed the superior effect of crocin coupled with AmpB (P < 0.0001) on promastigotes than crocin or AmpB (P < 0.001) alone relative to the untreated group.
On murine cell lines and DMEM culture media, various drug concentrations (0-200 μM) were applied, and the CC 50 rates of drugs were calculated using the counting means of intracellular amastigotes.Examination of cytotoxicity at expected drug concentrations indicated that the drugs did not have a lethal effect, as the safety index (SI), which is calculated as CC50/ IC50, was found to be within acceptable limits, with an SI value of at least 1.The selectivity index (SI) for AmpB, crocin, and crocin plus AmpB was 7.5, 20.2, and 18.1, respectively (Table 3).

Effect of crocin, AmpB, and combination on amastigotes
The study observed significant differences in the indices of L. major intra-cellular amastigotes when compared to the negative group (P < 0.001).Table 3 provides the values for IC50, CC50, and the selectivity index (SI) of crocin, AmpB, and the combination of both.We determined that the CI index was 0.81 which is defined as synergism (CI<1) and our analysis verified that theoretical IC 50 was 69.75 μM had a significant difference from experimental IC 50 was 24.5 μM (P< 0.001) that represented a synergistic effect in our combination activity (Fig 3).
The activity of crocin and AmpB alone were comparable; however, at 6.25 μM, crocin did not exhibit any response in killing the intracellular amastigotes (Table 4).However, the  crocin/AmpB mixture effect was enhanced, and at a 200 μM combination, no amastigotes were alive (P < 0.001) (Table 5).Fig 4 exhibits the mean mortality rates of L. major promastigotes (nonclinical stage) treated with various concentrations of crocin, AmpB, or both.The results demonstrated that crocin and AmpB alone were meaningfully effective (P < 0.001) against L. major promastigotes, but their action was significantly improved (P < 0.0001) in combination.

Effect of crocin, AmpB, or combination on arginase activity
The outcome presented that by increasing the concentration of crocin, AmpB, and a combination of them, ARG activity levels of treated macrophages significantly decreased (P < 0.001) relative to the untreated control group (Fig 5).

Effect of crocin, AmpB, and combination on the gene expression profile
Comparison of cytokines expression of Th1 cytokines (IFN-γ, IL-12p40, and TNF-α) (Fig 6 ), iNOS, and STAT1, c-Fos, and Elk-1 (Fig 7) displayed elevated levels in treated macrophages compared untreated control group.On the other hand, IL-4, IL-10, and TGF-β gene expression (as a marker of the Th2 pathway) were reduced by increasing drug concentrations (Fig 8).The gene expression profiles in Th1 and Th2 phenotypes and transcription factors in the crocin and AmpB alone were the same.However, a significant upsurge of Th1 line and transcription genes and a substantial diminution in the Th2 subset at comparable concentrations were detected (P < 0.001).

Effect of crocin, AmpB, and combination on NO generation
The results presented that crocin and AmpB alone or in combination could significantly increase nitrite production in macrophages infected by L. major by increasing drug concentrations relative to the untreated control group (Fig 9).While at lower concentrations of crocin (12.5 μM and 25 μM), AmpB (12.5 μM or combination (12.5+12.5 μM), no effect was observed.

Effect of crocin, AmpB, and combination on ROS production
Crocin, AmpB alone, or in combination similarly promoted the ROS level (P < 0.001) in the treated intra-macrophage amastigotes following a dose-response profile compared to untreated control (Fig 10).

Effect of crocin, AmpB, and combination on programmed cell death in L. major
Different concentrations of crocin, AmpB, or combination significantly induced the apoptotic profiles compare untreated control group (P < 0.001), except crocin alone at 12.5 μM showed

Discussion
Existing drugs used to treat leishmaniasis include antimonials, AmpB, miltefosine, allopurinol, pentamidine, and azole compounds [34].However, these drugs are often ineffective, and expensive, associated with the emergence of drug resistance.The desperate need for alternative therapeutics or the co-administration of other therapeutics is crucial.Natural products and phytomedicines have represented a well-established and valued source of potentially bioactive compounds in drug discovery, resulting in a durable interest in developing natural products to combat all forms of leishmaniasis [35,36].This complex disease is endemic in low-income countries with little incentive for pharmaceutical companies to participate in developing new drugs.Without proper medical and health infrastructures, people in these areas increasingly appreciate natural medicines as therapeutics.This study showed that crocin established an antileishmanial activity.However, the lethal effect was more significantly enhanced when combined with AmpB.The higher efficiency of the crocin and AmpB mixture in hindering the propagation of L. major in macrophage assays was mediated by elevating immune elements and preventing Leishmania arginase (L-ARG) levels.The Leishmania arginase activity has focused on intense investigations [36].L-ARG is the primary enzyme in Leishmania polyamine biosynthesis.Recent studies have revealed the significance of polyamines for Leishmania persistence, growth, differentiation, and infectivity and corroborated this biochemical pathway as a critical therapeutic drug target [37].The current results displayed that the cell treated with different crocin/AmpB mixture concentrations inhibited L-ARG uptake and enhanced the parasite killing.This finding is consistent with nicotinamide inhibition of L-ARG on L. tropica, the causative agent of urban CL [38].
Arginine is also the precursor in the biosynthesis of many proteins and nitric oxide (NO).Hence, arginine is crucial to produce NO in phagocytic cells through iNOS potentiation of the immune response, contributing to parasite death [39].Crocin can trigger adaptive immune functions via T CD + 4 lymphocytes associated-cytokine production, a prominent Th1 phenotype cell line polarization towards the significant immune expression of cytokines including TNF-α, IFN-, iNOS, and allied transfer factors (Stat-1, c-Fos, and Elk-1).Recovery from CL typically depends on introducing T-cell proliferation, predominantly Th1 response, primed by IL-12p40, dendritic cells (DCs), and macrophages [40].IFN-produced from IL-12p40 signed T-lymphocytes stimulate tumor necrosis factor (TNF)-α and NO-mediated alleviation of the organisms [41].Similar to the potentiation of IL-12p40 and supplementary pro-inflammatory cytokines, expression levels of Th2 cytokines have likewise been broadly explored [42].IL-4, IL-10, and TGF-β prevent the production of IFN-γ released from macrophages.IL-4 is recognized to inhibit macrophage stimulation effectively, but IL-10 plays a pivotal role in CL evolution.
Modulating immune responses with natural remedies and secondary products has been confirmed as a promising therapeutic approach [43,44].Approximately 80% of the global public still uses herbal medicine for their clinical and healthcare requirements [38,45].World Health Organization (WHO) rationalized its Traditional Medicine Strategy for 2014-2023 to promote medicinal plants [46,47].The strategy aims to support the countries in developing proactive campaigns and reinforce traditional medicine's role in keeping populations healthy.The immunostimulatory phytochemicals are the primary and rational basis of potential leishmanicidal and may provide new strategies to combat leishmaniasis, alone or as a combination.Many plant-extracted macromolecules revealed strong properties on immune system roles in experimental models and signified their beneficial potential [38,[48][49][50].
The chief active derivative of saffron is crocin [51].Crocin, a saffron glycoside, is a carotenoid having four analogs: crocin 1, crocin 2, crocin 3, and crocin.Reports have indicated that saffron possessed practical value with numerous pharmacological properties, including antioxidant, anti-inflammatory, and cardioprotective effects.Some studies showed that crocin protects against many degenerative chronic diseases [52,53].A study revealed that the oral treatment of crocin within one month in healthy volunteers was safe compared with the control group [54].
In the life cycle, leishmania parasites infect mammalian macrophages where the internalization of organisms triggers substantial quantities of ROS mediates to control infection, as they are significantly expressed in this study.The mechanism of action of different antileishmanial active ingredients, including crocin/AmpB, involves the generation of ROS to enable the killing process of the organism.High volumes of ROS are fatal to promastigote and amastigote forms of Leishmania donovani.A study showed that L. donovani responsible promastigote stage undergoes PCD on treatment with H 2 O 2 [55].High levels of ROS are reported to be a deadly weapon exerted by phagocytic cells for impairment of critical cellular organic substances like proteins, lipids, and DNA, thus resulting in apoptosis of the causative agent [56,57].ROS in leishmanial agents could be induced due to cellular and drug uptake [58].Various chemotherapeutic compounds used against Leishmania species or cancer treatment facilitate their effects by generating ROS [59][60][61][62][63].The crucial docking technique was devised to anticipate drug-receptor interactions due to the strong crocin and NO affinity and the formation of large quantities of oxidative metabolites [27].
Considering the fundamental biology of Leishmania species requires thoughtful of the cell cycle.We have established that L. major undergoes significant changes in ultrastructural profile [64].Different cell cycle phases may be seen in a broad spectrum of microstructural features found in cultures during exponential development.Current data displayed that treatment with crocin/AmpB perturbed the Leishmania cell cycle growth, or mitosis, and promoted DNA synthesis.Induced cell propagation arrest utilizes natural components to stop progression through the cell cycle.This feature could be an indicator for evaluating and monitoring the experimental drugs' action.
The leishmanicidal effect of crocin/AmpB was also mediated through apoptotic-like effects as evidenced by phosphatidylserine (PS) externalization [49,65].This significant change appears due to reduced phospholipids translocase activity and activation of a calcium-dependent scramblase.Exposure of PS on the exterior membrane of the cells is an apparent change common to various apoptotic cells and cell-cycle arrest at different stages of the growth phase.Generally, these findings indicate that crocin/AmpB has a promising antileishmanial effect facilitated by programmed cell death and further advanced study as a possible therapeutic choice for the treatment of leishmaniasis.
Antimonials have been changed with AmpB per the instructions for treating leishmaniasis.AmpB is the second-line treatment for antimony strains that have developed resistance [66].This polyene antifungal drug attaches to ergosterol to target the cell walls of promastigotes and amastigotes [67,68].Leishmaniasis is one of several diseases for which AmpB has been employed for decades as an authorized medicine.AmpB might work in concert with paromomycin or miltefosine to reduce the extracellular promastigotes, suppress intracellular amastigotes, and restrict the disease over time [69].Combining plant immunomodulators with traditional medications like AmpB may enable the effective treatment of a variety of molecular targets, improving therapeutic effectiveness and reducing toxicity [70].
Liposomal AmpB has been used extensively to treat VL due to its higher safety and efficacy profile [71].It is the standard drug for immunocompetent patients, and the combination treatment is an acceptable choice because of the likely chemical achievement of two medicines in lower dosages with different mechanistic actions.The rationale behind combination or polytherapy is to increase activity by using compounds with synergistic or additive interaction.This approach has increasingly been supported to upsurge treatment efficacy, reduce treatment period/cost, and delay or halt the emergence of recrudescence and resistance [72].Based on crocin's antileishmanial effect and targeting to detect potential new therapeutic alternatives for leishmaniasis treatment, this study was accomplished to evaluate the activity of crocin in combination with AmpB as a conventional antileishmanial drug.The outcomes are very encouraging and may further support the justification of combination therapy for CL to provide safe and effective options.
In conclusion, this study presented the highest effect of the crocin/AmpB combination in hindering the multiplication of L. major stages in a macrophage assay by inhibiting L-ARG levels, potentiating immune response, and arresting cell cycle growth.Therefore, with multiple mechanistic actions, a high safety index on mammalian cells, and potent antioxidative activity combined with AmpB, crocin could be esteemed as a basis for a potential bioactive component and a logical source for leishmanicidal drug development against CL in future advanced clinical settings.

Fig 1 .
Fig 1. Docking.A) Nitric oxide (NO) consists of a central pocket and 4 cavities.B) Predicted amino acids in pocket formation by PLIP web tool.C) Crocin binds to NO with the active site residues by LIGPLOT program.D) Molecular docking by Molgro Virtual Docker software.https://doi.org/10.1371/journal.pone.0291322.g001

Table 3 . Evaluating the IC 50 values of crocin and crocin plus amphotericin B (AmpB) against amastigotes and promastigotes forms of L. major compared with AmpB and CC 50 values of the drugs on macrophages using the SI index.
c SI; Selectivity index (CC 50 /IC 50 ).NR: Not related.https://doi.org/10.1371/journal.pone.0291322.t003